Identification of Pathways for Production of d-Glucaric Acid by Pseudogluconobacter saccharoketogenes

Pseudogluconobacter saccharoketogenes produces glucaric acid from d -glucose via two pathways, i.e., through d -glucuronic acid or d -gluconic acid. These pathways are catalyzed by alcohol dehydrogenase, aldehyde dehydrogenase, and gluconate dehydrogenase. Although d -glucaraldehyde and l -guluronic acid are also theorized to be produced in pathways throsugh d -glucuronic acid and d -gluconic acid, respectively, no direct data to identify these intermediates have been reported. In this study, the intermediates were purified and identified as d -glucaraldehyde and l -guluronic acid. The substrate specificities of the three enzymes on these intermediates and their oxidation products were studied, and the roles of alcohol, aldehyde, and gluconate dehydrogenases in d -glucaric acid-producing pathways were elucidated using the intermediates. Additionally, the substrate specificities of alcohol and aldehyde dehydrogenases on some alcohols, aldehydes, and aldoses were determined. Alcohol dehydrogenase showed wide substrate specificities, whereas the substrates oxidized by aldehyde dehydrogenase were limited. A 30-L scale reaction using the resting cells of Rh47-3 revealed that d -glucaric acid was produced from d -glucose and d -gluconic acid in 60.3 mol% (7.0 g/L) and 78.6 mol% (22.5 g/L) yields, respectively.